FIGURE 3.

MITF mRNA is a target of miR-340. A, 451Lu cells were co-transfected with Tet-off plasmid, p-BIG-MITF plasmid with short 3′-UTR, and the indicated miR-Sponge construct. Transcription was stopped by treatment with doxycycline for the indicated durations. The stability of MITF transcripts was analyzed by measuring MITF mRNA levels with real time RT-PCR (normalized to GAPDH expression). B, 451Lu cells were co-transfected with Tet-off plasmid, p-BIGL-MITF-Short MITF 3′-UTR, and the indicated miR-Sponge construct. The turnover of chimeric Luciferase-MITF-3′-UTR transcripts was analyzed as in A. C, 451Lu cells were co-transfected with β-galactosidase-expressing plasmid, Tet-off plasmid, p-BIGL-MITF^wt, or p-BIGL-MITF^δmiR-340 and the indicated miR-Sponge constructs. After 24 h the luciferase activity was measured. The values represent luciferase activity normalized to β-galactosidase. D, 451Lu cells were co-transfected with Tet-off plasmid, p-BIG-MITF^wt, or p-BIG-MITF^δmiR-340. The stability of MITF transcripts was analyzed as in A. All of the results are representative of three separate experiments and are expressed as the mean values ± S.D. (error bars). The average half-lives of mRNAs are presented in supplemental Table S3. The levels of endogenous miR-340 are presented in supplemental Fig. S1A.